JP5436225B2 - New sialidase - Google Patents

Description

Detailed Description of the Invention

[Field of the Invention]
The present invention relates to a novel sialidase.

[Background of the invention]
Sialic acid comprises a family of about 40 derivatives of the 9 carbon sugar neuraminic acid. It is a strong organic acid with about 2.2 pK _a. Unsubstituted neuraminic acid does not exist in nature. The amino group is usually acetylated to yield the most popular form of sialic acid, N-acetylneuraminic acid, although other forms exist (Traving et al. Cell Mol Life Sci (1998) 54, 1330-1349). Sialic acids are found in the animal kingdom, from echinoderms to humans, while their presence in lower animals or plants of the anterior mouth strain is not suggested. The only known exception is the presence of polysialic acid in the insect Drosophila larvae. In addition, sialic acid is also found in some protozoa, viruses and bacteria. The sialoglycoconjugate is present on the cell surface as well as on the intracellular membrane. In higher animals they are also important components of serum and mucosal material.

  Sialic acid has a variety of biological functions. Due to their negative charge, sialic acids are involved in the binding and transport of positively charged molecules such as calcium ions, and the attraction and repulsion phenomenon between cells and molecules. In addition to their size and negative charge, the fact that they are located at the end of the exposed carbohydrate chain allows them to function as a protective shield at the sub-terminal end of the molecule or cell. They can prevent, for example, glycol-proteins from being degraded by proteases, or can prevent respiratory mucosal layers from bacterial infection. An interesting phenomenon is the spreading effect exerted on sialic acid-containing molecules by the repulsive force acting between their negative charges. This stabilizes the exact conformation of the enzyme or membrane (glyco) protein and is important for the slimy nature and the sliding and protective function of mucosal substances such as the ocular surface and mucosal epithelium ( Traving et al. Cell Mol Life Sci (1998) 54, 1330-1349). Clearly, treatment of such sialic acid-containing materials with an appropriate sialidase can dramatically affect the biological and physical characteristics of such materials. Treatment of sialic acid-containing proteins with sialidase can make them more susceptible to degradation by proteases, and treatment of mucosal material with sialidase can strongly reduce or eliminate their mucosal characteristics. Such changes are interesting when such proteins require an industrial process of processing (eg, proteolysis), eg, for protein hydrolysates.

  Sialic acids are involved in a variety of recognition processes between cells and molecules. Therefore, the immune system can distinguish between self-structures and non-self structures according to their sialic acid patterns. Sugar represents antigenic determinants such as blood group substances and is a necessary component of the receptor for many endogenous substances such as hormones and cytokines. In addition, many virulence factors such as toxins (eg, cholera toxin), viruses (eg, influenza), bacteria (eg, Escherichia coli, Helicobacter pylori) and protozoa (eg, trypanosoma) Trypanosomes cruzi also binds to host cells via sialic acid-containing receptors, another important group of sialic acid recognition molecules is plants, animals and invertebrates that bind to specific sugar residues It belongs to the lectin, which is a normal oligomeric glycoprotein derived from, for example, wheat germ agglutinin, Limulus polyphemus aagglutinin, Sambucus nigr a) Agglutinin and Maackia amurensis agglutinin These lectins appear to aid plants in their defense against sialic acid-containing microorganisms or herbivorous mammals. Siglec is included (Traving et al. Cell Mol Life Sci (1998) 54, 1330-1349) and has a variety of physiological roles.Sialic acid can also assist in cellular and molecular shielding. The red blood cells are covered with a protective layer of sialic acid molecules, which are stepped out during the life cycle of the blood cell, and then the penultimate galactose residue representing the signal for degradation Exposed, then (than), shielded Untreated blood cells bind to macrophages and are phagocytosed Some other examples of such shielding strategies are known, which can also have a detrimental effect, which corresponds to It can be seen from some of the tumors that are sialicated to a much higher degree than the tissue, and as a result, the masked cells are not exposed to the immune defense system and high sialic acid content is also associated with further cell proliferation The shielding effect of sialic acid also helps to conceal antigenic sites on the parasite cells and keeps them from being exposed to the system. This is the case with microbial species such as E. coli strains and Neisseria gonorrhoeae, where treatment of such species with sialidase hides them from the immune system. Affects those possibilities.

  Sialidase (neuraminidase, EC 3.2.1.18) hydrolyzes terminal non-reducing sialic acid bonds in glycoproteins, glycolipids, gangliosides, polysaccharides and synthetic molecules. Sialidases, called transsialidases, can also carry out transfer reactions that transfer sialic acid residues from one molecule to another. Sialidases are commonly found in animals of the new mouth animal lineage (Echinodermata to Mammalia) and also in the diverse microorganisms that are most abundant as animal symbiotic or pathogens. Sialidases and their sialyl substrates appear to be absent from plants and most other metazoans. Even among bacteria, sialidases are found irregularly such that this property differs even in related species or strains of one species. Sialidases have also been found in viruses and protozoa (Traving et al. Cell Mol Life Sci (1998) 54, 1330-1349). Microorganisms containing sialidase often survive in contact with higher animals as hosts, for example, as parasites. Here, they may have trophic functions, allowing their owners to scavenge the host sialic acid for use as a carbon source. In some microbial pathogens, sialidase may act as a virulence factor. The role of salidases as a factor in pathogenicity is controversial. On the one hand, they support the influence of pathogenic microbial species such as Clostridium perfringens. On the other hand, these enzymes are common factors for carbohydrate catabolism in many non-pathogenic species, including higher animals. However, they do not exert a direct toxic effect (Traving et al. Cell Mol Life Sci (1998) 54, 1330-1349). Instead, their detrimental effects depend on the large amount of enzyme released into the host along with other toxic factors upon induction by host sialic acid under non-physiological conditions.

  Mammalian sialidases are usually about 40-45 kD in size. There have been no reports of attempts to overexpress mammalian sialidase and produce it to an amount of industrial interest. The human sialidase can be a lysosomal enzyme, a cytosolic enzyme, or a membrane-bound enzyme (Achiyutan and Achiyutan (2001) Comp. Biochem. Phys. B, 129, 29-64). Lysosomal sialidase is a glycosylation enzyme. Sialidases contain a conserved motif. The most prominent conserved motif is the so-called Asp box, which is a stretch of amino acids of the general formula —S—X—D—X—G—X—T—W—, where X represents a variable residue It is. With the exception of viral sialidase, where this motif is found only once or twice or even not present, this motif is found 4-5 times throughout all microbial sequences. The third Asp box is more conserved than Asp boxes 2 and 4. The space between two consecutive Asp boxes is also conserved between different primary structures (Traving et al. Cell Mol Life Sci (1998) 54, 1330-1349). The Asp box probably plays a structural role and is probably not involved in the catalyst. In contrast to the Asp box, the FRIP-motif is located in the N-terminal part of the amino acid sequence. It includes the amino acid —X—R—X—P— with absolutely conserved arginine and praline residues. Arginine is directly involved in the catalyst through the binding of substrate molecules. The glutamate-rich region between asp boxes 3 and 4, as well as two additional arginine residues, are also important for catalysis (Traving et al. Cell Mol Life Sci (1998) 54, 1330-1349).

  Microbial sialidases can be classified according to their size into two groups: a small protein of about 42 kDa and a large protein of 60-70 kDa. The primary structure of a large sialidase contains an additional stretch of amino acids between the N-terminus and the second Asp box and between the fifth Asp box and the C-terminus. They are thought to contribute to the broader substrate specificity of large sialidases. Similar to mammalian sialidase, the bacterial counterpart contains an F / YRIP motif and some Asp box. Bacterial sialidases are often associated with mucosal infection and virulence. For this reason, the larger bacterial sialidase has not been recognized as suitable for use as a processing aid in food or pharmaceutical applications. Small sialidases (same size as mammalian sialidases) have been identified in the bacteria shown above. That is, Clostridium perfringens contains a small sialidase with a size of about 40 kDa without an extension common to sialidases of other bacteria. However, this Clostridium sialidase is not secreted by bacteria and is therefore not involved in virulence (Rogggentin et al. (1995) Biol Chem Hope Seyler 376, 569-575). It is of great interest that only bacterial sialidases with additional extensions are involved in virulence. Overexpression of bacterial sialidase in E. coli generally results in low production rates; small Clostridium sialidase is only produced up to 1 mg / l as an intracellular protein in E. coli. (Kruse et al. (1996) Protein Expr Purif. 7, 415-422).

  Uchida et al. (Biochimica et Biophysica Acta, Vol. 350, No. 2, 1974, pages 425-431) describe screening for microbial neuraminidase induced by colominic acid. Of the 1000 microorganisms screened, Sporotrichium schenckii, Penicillium urticae and Streptomyces sp. Neuraminidase was obtained. Since Penicillium urticae is a fungus involved in food spoilage, it is not a suitable production organism for food-grade sialidase, and Sporotrichium schenckii is a pathogenic fungus is there. The species name in the bacteria Streptomyces sp has not been determined. The MW of the sialidase described in this paper is unknown. Iwamori et al. (J. Biochem. 138, pp. 327-334) disclose the bacterium Arthrobacter urefaciens sialidase. However, this bacterial neuraminidase is known to be associated with HIV-1 mediated syncytium formation and virus binding / entry processes (Sun et al., Virology 284, 26-36, 2001). Thus, there is clearly a need for small non-virulent sialidases that are well produced for use in food and pharmaceuticals.

  In particular, the findings of secreted fungal sialidase are beneficial because secreted enzymes are easily overexpressed and can be purified in large quantities from fungal cultures. This dramatically reduces the cost for the production of sialidase.

[Summary of Invention]
The present invention relates to an isolated polypeptide having sialidase activity selected from the group consisting of:
(A) a polypeptide having an amino acid sequence having at least 60% amino acid sequence identity with amino acids 1 to 407 of SEQ ID NO: 3;
(B) at low stringency conditions, (i) at least 80% or 90% identical over 60 nucleotides, preferably over 100 nucleotides, more preferably at least 90% identical over 200 nucleotides. Or (ii) a polypeptide encoded by a polynucleotide that hybridizes with a nucleic acid sequence complementary to the nucleic acid sequence of SEQ ID NO: 1 or 2. Preferably, the polypeptide has a MW (molecular weight) (SDS-page) of less than 55 kDa, more preferably the polypeptide has a MW (molecular weight) (SDS-page) of less than 52 kDa, or The polypeptide has a MW (molecular weight) of less than 50 kDa (calculated based on amino acid sequence), more preferably, the polypeptide has a MW (molecular weight) of less than 45 kDa (calculated based on amino acid sequence). Have.

  Furthermore, the present invention provides polypeptides that have sialidase activity and are non-virulent. Also, part of the present invention is a polypeptide having sialidase activity, said polypeptide being a fungal sialidase, having a MW (molecular weight) (SDS-Page) of less than 55 kDa, more preferably this fungus The sialidase has a MW (molecular weight) (SDS-Page) of less than 52 kDa, or the fungal sialidase has a MW (molecular weight) (calculated based on amino acid sequence) of less than 50 kDa, more preferably the fungus Sialidase has a MW (molecular weight) (calculated based on amino acid sequence) of less than 45 kDa.

  Preferably, the polypeptide of the invention is at least 65%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% of amino acids 1 to 407 of SEQ ID NO: 3. And most preferably having an amino acid sequence having at least about 97% identity. The invention also encodes a polypeptide of claim 1 or under low stringency conditions, more preferably under moderate stringency conditions, and most preferably high stringency conditions. It relates to an isolated polynucleotide comprising a nucleic acid sequence that hybridizes under conditions to SEQ ID NO: 1 or 2. In addition, the present invention discloses a nucleic acid construct comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of the polypeptide in a suitable expression host. Furthermore, the present invention provides a recombinant expression vector comprising this nucleic acid construct and a recombinant host cell comprising said nucleic acid construct. According to another aspect of the invention, culturing the above strain / recombinant host cell to produce a supernatant and / or cell comprising the polypeptide; and recovering the polypeptide. A method for producing a polypeptide is disclosed. According to a further aspect of the invention, culturing a host cell comprising a nucleic acid construct comprising a polynucleotide encoding a polypeptide under conditions suitable for production of the polypeptide; and recovering the polypeptide Disclosed is a method for producing a polypeptide of the invention comprising: A polypeptide of the invention having sialidase activity is advantageously non-virulent. The polypeptides of the present invention can be used in the preparation of food or feed or as a pharmaceutical or part of a pharmaceutical.

Detailed Description of the Invention
According to the present invention, preferably a small sialidase with a MW (Molecular Weight) (SDS-Page) of less than 55 kDa (more preferably, the polypeptide has a MW (Molecular Weight) (SDS-Page) of less than 52 kDa) Or having a MW (molecular weight) (calculated based on amino acid sequence) of less than 50 kDa (more preferably, the polypeptide has a MW (molecular weight) (calculated based on amino acid sequence) of less than 45 kDa ) Is disclosed. Advantageously, the sialidase, extracellular enzyme of the present invention. Furthermore, the sialidase is preferably derived from a fungus. In general, the polypeptides of the present invention have a MW (molecular weight) (SDS-Page) greater than 35 kDa, more preferably the polypeptide has a MW (molecular weight) (SDS-Page) greater than 40 kDa, or The polypeptide has a MW (molecular weight) greater than 35 kDa (calculated based on amino acid sequence), more preferably the polypeptide has a MW (molecular weight) greater than 40 kDa (calculated based on amino acid sequence). Have

  An exoenzyme, or extracellular enzyme, is an enzyme that is secreted from the cell and acts outside the cell. It is used to break down large molecules that cannot enter cells without breaking down. Enzymes secreted by the organism into the environment act outside the microorganism. The antonym of exoenzyme is called endo-enzyme or intracellular enzyme (source is Wikipedia).

  In our theory, large sialidases are generally associated with mucosal infection and virulence and are therefore not suitable in food or pharmaceutical applications. In contrast, small sialidases are considered suitable for food or pharmaceutical applications.

  However, the present invention does not affirm or deny the accuracy of this theory. The present invention relates to an extracellular sialidase having a MW (molecular weight) (SDS-Page) of less than 55 kDa (more preferably, the sialidase has a MW (molecular weight) (SDS-Page) of less than 52 kDa), or a MW of less than 50 kDa Provided is an extracellular sialidase (more preferably, the sialidase has a MW (molecular weight) (calculated based on amino acid sequence) of less than 45 kDa (molecular weight) (calculated based on amino acid sequence).

  The present invention relates to a new sialidase identified in the fungus Penicillium chrysogenum.

  Advantageously, the present invention meets the need for sialidases that can be produced in large quantities. Preferably, such sialidase is secreted from the host cell. Active secretion is most important for an economical production process because it allows the recovery of almost pure forms of the enzyme without experiencing tedious purification processes. Overexpression of such actively secreted sialidase by a food grade fungal host such as Aspergillus produces a food grade enzyme and a cost effective production process and is therefore preferred. This secretory sialidase was first found in filamentous fungi. Disclosed is a process for the production of large quantities of sialidase by the food grade production host Aspergillus niger.

  From an economic point of view, there is clearly a need for improved methods of producing sialidase in large quantities and in a relatively pure form compared to low production rates of mammalian and bacterial sialidases. A preferred way to do this is through the overproduction of such sialidases using recombinant DNA technology. A particularly preferred way to do this is through the overproduction of fungal sialidase, and the most preferred way to do this is through the overproduction of Penicillium sialidase. . In order to enable the latter production pathway, unique sequence information of Penicillium-derived sialidase is essential. More preferably, the entire nucleotide sequence of the coding gene must be available.

  An improved means of producing the newly identified secreted sialidase in abundant and relatively pure form is through the overproduction of the enzyme encoded by Penicillium using recombinant DNA technology. A preferred way to do this is through the overproduction of such secreted sialidases in food grade host microorganisms. Well-known food-grade microorganisms include Aspergilli, Trichoderma, Streptomyces, Bacilli, and Saccharomyces and Kluyveromyces yeasts such as Kluyveromyces. An even more preferred way to do this is through overproduction of secreted Penicillium-derived sialidase in food grade fungi such as Aspergillus. Most preferred is overproduction of secreted sialidase in food-grade fungi in which the codon usage of the gene encoding sialidase is optimized for the food-grade expression host used. In general, unique sequence information for secreted sialidase is desirable to enable the latter optimization pathway. More preferably, the entire nucleotide sequence of the sialidase coding gene must be available. Once the gene encoded by the secreted sialidase is transformed in a suitable host, the secreted strain can be used for fermentation and isolation of the secreted sialidase protein from the fermentation broth.

  Once the new enzyme becomes available in large quantities and relatively pure food, foodstuffs (such as cheese), protein hydrolysates with improved texture and / or immunological properties are Can be prepared in a grade and economical manner. Enzymes can also potentially be used as preservatives. By removing protective sialic acid residues from the bacterial wall, microorganisms are recognized and eliminated by the immune system.

  The polypeptide of the present invention having sialidase activity may be in an isolated form. As defined herein, an isolated polypeptide is essentially free of other non-sialidase polypeptides and typically is at least about 20% as determined by SDS-PAGE. Pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, even more preferably about 90% pure, and most preferably about 95% pure, Produced or recombinant polypeptide. Polypeptides may be isolated by centrifugation and chromatographic methods, or any other technique for obtaining pure protein from a crude solution. It will be appreciated that the polypeptide may be admixed with a carrier or diluent that does not interfere with the intended purpose of the polypeptide, and thus this form of the polypeptide will still be recognized as isolated. It generally comprises a polypeptide in the preparation, wherein the polypeptide of the invention is more than 20%, eg 30%, 40%, 50%, by weight of the protein in the preparation. %, 80%, 90%, 95% or over 99%.

  Preferably, the polypeptide of the present invention can be obtained from a microorganism possessing a gene encoding an enzyme with sialidase activity. More preferably, the polypeptide of the present invention is secreted from a microorganism. Even more preferably, the microorganism is a fungus and optionally a filamentous fungus. Therefore, a preferred donor organism is a genus Penicillium such as the species Penicillium chrysogenum. In a first embodiment, the invention provides at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably with respect to amino acids 1 to 407 of SEQ ID NO: 3 (ie, polypeptide). Provided is an isolated polypeptide having an amino acid sequence identity with at least 90%, even more preferably at least 95%, and most preferably at least 97% amino acid sequence identity and having sialidase activity.

  For the purposes of the present invention, the degree of identity between two or more amino acid sequences is determined by the BLAST P protein database search program (Altschul et al., 1997, Nucleic Acids) with matrix Blosum62 and 10 predicted thresholds. Research 25: 3389-3402).

  The polypeptide of the invention may comprise the amino acid sequence set forth in SEQ ID NO: 3, or a substantially homologous sequence, or a fragment of any sequence having sialidase activity. In general, the naturally occurring amino acid sequence shown in SEQ ID NO: 3 is preferred.

  One aspect of the present invention is a polypeptide having the amino acid sequence shown in SEQ ID NO: 3, whereby the signal sequence is removed. Secreted enzymes, such as the amino acid sequence of SEQ ID NO: 3 containing pre- or signal-sequences and / or pro-sequences, are often synthesized. These sequences are often removed from the protein either during or after the secretion process. Thus, mature secreted proteins often no longer contain these pre- and pro-sequences. Thus, a polypeptide having an amino acid sequence having at least 60% amino acid sequence identity with amino acids 1 to 407 of SEQ ID NO: 3 and lacking a possible pre- or pro-sequence is part of the present invention. A possible processing site in the amino acid sequence of SEQ ID NO: 3 is behind amino acid 33. In this case, the mature enzyme according to the invention starts at amino acid number 34. Other modifications from the amino acid sequence of SEQ ID NO: 3 by further processing such as amino acid removal are permissible as long as they do not interfere with the activity of the enzyme.

  The polypeptide of the invention may also comprise a naturally occurring variant or species homologue of the polypeptide of SEQ ID NO: 3.

  A variant is a polypeptide that occurs naturally in, for example, a fungus, bacterium, yeast or plant cell, which variant has a sequence substantially similar to the sialidase activity and the protein of SEQ ID NO: 3. The term “variant” refers to a polypeptide having the same essential characteristics or basic biological function as the sialidase of SEQ ID NO: 3, and includes allelic variants. Preferably, the mutant polypeptide has at least the same level of sialidase activity as the polypeptide of SEQ ID NO: 3. Variants include allelic variants derived from either the same strain as the polypeptide of SEQ ID NO: 3 or a different strain of the same genus or species.

  Similarly, a species homologue of a protein of the invention is an equivalent protein of similar sequence that is a sialidase and occurs naturally in another species.

  Variants and species homologues are isolated using the procedures described herein and performing such procedures on appropriate cell sources, such as bacterial, yeast, fungal or plant cells. can do. In addition, in order to obtain a clone expressing a variant or species homologue of the polypeptide of SEQ ID NO: 3, a DNA library prepared from yeast, bacteria, fungi or plant cells is searched using the probe of the present invention. It is also possible to do. Methods that can be used to isolate known gene variants and species homologues are extensively described in the literature and are known to those skilled in the art. These genes can be manipulated by conventional techniques to produce the polypeptides of the present invention and then produced by recombinant or synthetic techniques known per se.

  The sequence of the polypeptide of SEQ ID NO: 3 and variants and species homologues can also be modified to provide the polypeptides of the invention. Amino acid substitutions can be made, for example, from 1, 2, or 3-10, 20 or 30 substitutions. The same number of deletions and insertions may be made. These changes may be made outside of a region critical to the function of the polypeptide, such that the modified polypeptide retains its sialidase activity.

  The polypeptide of the present invention includes fragments of the above-mentioned full-length polypeptide and variants thereof, and includes a fragment of the sequence set forth in SEQ ID NO: 3. Such a fragment typically retains activity as a sialidase. Fragments may be at least 50, 100 or 200 amino acids long, or such number of amino acids shorter than the full length sequence shown in SEQ ID NO: 3.

  Usually, the polypeptides of the invention are produced recombinantly as described below, but they can also be produced by synthetic means if necessary. Synthetic and recombinant polypeptides are modified, for example, by the addition of histidine residues or T7 tags to aid their identification or purification, or by the addition of signal sequences to facilitate their secretion from the cell. obtain.

  Thus, the mutant sequence may comprise a sequence derived from a strain of Penicillium other than the strain from which the polypeptide of SEQ ID NO: 3 was isolated. Variants can be identified from other Penicillium strains by examining sialidase activity and cloning and sequencing as described herein. As long as the peptide maintains the basic biological function of the sialidase of SEQ ID NO: 3, the variant may comprise a deletion, modification or addition of a single amino acid or group of amino acids within the protein sequence.

  Amino acid substitutions can be made, for example, from 1, 2, or 3-10, 20 or 30 substitutions. The modified polypeptide generally retains activity as a sialidase. Conservative substitutions may be made; such substitutions are well known in the art.

  Shorter or longer polypeptide sequences are also within the scope of the invention. For example, as long as a peptide of at least 50 amino acids or 100, 150, 200, 300, 400, 500, 600, 700 or 800 amino acids in length demonstrates the basic biological function of the sialidase of SEQ ID NO: 3, This is considered to be within the scope of the present invention. In particular, but not exclusively, this aspect of the invention encompasses situations where the protein is a fragment of the complete protein sequence.

  The present invention also relates to a polynucleotide encoding a polypeptide having sialidase activity, wherein the polynucleotide comprises a polynucleotide sequence encoding amino acid SEQ ID NO: 3.

  In the present invention, it is particularly relevant that the protein of interest is actively secreted into the growth medium. Secreted proteins are usually synthesized initially as a pre-protein, and then the pre-sequence (signal sequence) is removed during the secretion process. The secretion process is basically similar in prokaryotes and eukaryotes: actively secreted pre-proteins pass through the membrane, signal sequences are removed by specific signal peptidases, and mature proteins are (re) folded Is done. In addition, the general structure of the signal sequence can be recognized. The signal sequence for secretion is located at the amino terminus of the pre-protein and is generally 15-35 amino acids long. The amino terminus preferably contains positively charged amino acids and preferably does not contain acidic amino acids. This positively charged region is believed to interact with the negatively charged head group of the membrane phospholipid. This region is followed by a hydrophobic transmembrane core region. This region is generally 10-20 amino acids long and consists primarily of hydrophobic amino acids. Charged amino acids are usually not present in this region. The transmembrane region is followed by a signal peptidase recognition site. The recognition site consists of amino acids that prefer small-X-small. The small amino acid can be alanine, glycine, serine or cysteine. X can be any amino acid. Using such rules, algorithms have been created that can recognize such signal sequences from eukaryotic and prokaryotic cells (Benttsen, Nielsen, von Heine ( von Heijne) and Brunak. (2004) J. Mol. Biol., 340: 783-795). A SignalP program for calculating and recognizing the signal sequence of proteins is generally available (http://www.cbs.dtu.dk/services/SignalP/).

  It is relevant to the present invention that the signal sequence can be recognized from the deduced protein sequence of the sequenced gene. If a gene encodes a protein whose signal sequence is predicted using the SignalP program, the protein is likely to be secreted.

  In a second embodiment, the present invention has sialidase activity and, under low stringency conditions, more preferably under moderate stringency conditions, and most preferably, high stringency conditions. (I) a nucleic acid sequence of SEQ ID NO: 1, or (ii) a nucleic acid fragment comprising at least a part of SEQ ID NO: 1, or (iii) a nucleic acid fragment having a base different from the base of SEQ ID NO: 1; Alternatively (iv) an isolated polypeptide encoded by a polynucleotide that hybridizes or is capable of hybridizing to a nucleic acid strand complementary to SEQ ID NO: 1.

  The term “capable of hybridizing” refers to a nucleic acid (eg, a nucleotide sequence of SEQ ID NO: 1, or a fragment thereof, or a complement of SEQ ID NO: 1 or a fragment thereof) in which the target polynucleotide of the present invention is used as a probe. And can be hybridized at a level significantly higher than the background. The invention also includes polynucleotides that encode the sialidases of the invention, as well as nucleotide sequences that are complementary thereto. The nucleotide sequence may be RNA or DNA, including genomic DNA, synthetic DNA or cDNA. Preferably the nucleotide sequence is DNA and most preferably genomic DNA sequence. Typically, a polynucleotide of the invention comprises a contiguous sequence of nucleotides that is capable of hybridizing under selective conditions to the coding sequence of SEQ ID NO: 1 or the complement of the coding sequence. Such nucleotides can be synthesized according to methods well known in the art.

The polynucleotide of the invention can hybridize to the coding sequence of SEQ ID NO: 2 or the complement of the coding sequence at a level significantly higher than background. For example, background hybridization can occur due to other cDNAs present in the cDNA library. The signal level produced by the interaction between the polynucleotide of the invention and the coding sequence of SEQ ID NO: 2 or the complement of the coding sequence is typically between other polynucleotides and the coding sequence of SEQ ID NO: 2. Strength of at least 10 times, preferably at least 20 times, more preferably at least 50 times, and even more preferably at least 100 times. The intensity of interaction can be measured, for example, by radiolabeling the probe, eg, with ³² P. Selective hybridization typically involves low stringency (0.3 M sodium chloride and 0.03 M sodium citrate, approximately 40 ° C.), moderate stringency (eg, 0.3 M sodium chloride and 0.03 M sodium citrate). Acid string, about 50 ° C.) or high stringency (eg, 0.3 M sodium chloride and 0.03 M sodium citrate, about 60 ° C.) can be used.

  The polynucleotide of the present invention also includes a synthetic gene capable of encoding the polypeptide of SEQ ID NO: 3 or a variant thereof. From time to time it is also suitable to adapt the codon usage of the gene to the preferred bias of the production host. Techniques for designing and constructing synthetic genes are generally available (ie, http://www.dnatwopointo.com/).

[Modify]
The polynucleotide of the present invention may comprise DNA or RNA. They may be single stranded or double stranded. They may also be polynucleotides comprising synthetic or modified nucleotides containing peptide nucleic acids within them. Many different types of modifications to polynucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and the addition of acridine or polylysine chains at the 3 'and / or 5' ends of the molecule. For the purposes of the present invention, it should be understood that the polynucleotides described herein can be modified by any method available in the art.

  Those skilled in the art will be able to use routine techniques to make any identification to express a polypeptide of the invention with nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotide of the invention. It should be understood that this can be reflected in the codon usage of the host organism.

  The coding sequence of SEQ ID NO: 2 may be modified by nucleotide substitutions, for example, 1, 2, or 3-10, 25, 50, 100, or more substitutions. The polynucleotide of SEQ ID NO: 2 can be alternatively or additionally modified by one or more insertions and / or deletions and / or by extensions at either or both ends. The modified polynucleotide generally encodes a polypeptide having sialidase activity. For example, as discussed below with respect to a polypeptide, when a modified sequence is translated, degenerate substitutions can be made and / or substitutions that result in conservative amino acid substitutions can be made.

[Homologue]
Nucleotide sequences that are capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO: 2 are included in the present invention and are generally at least 60, preferably at least 100, more than SEQ ID NO: 2. Preferably at least 50% or 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% with the coding sequence of SEQ ID NO: 2 over a region of at least 200 contiguous nucleotides, or most preferably over the entire length or Have at least 99% sequence identity. Similarly, nucleotides that encode active sialidase and are capable of selectively hybridizing to fragments of the complement of the DNA coding sequence of SEQ ID NO: 2 are also encompassed by the present invention.

  Any combination of the above degree of identity and minimum size may be used to define the polynucleotides of the invention, with more stringent combinations (ie higher identity over a longer length) Is preferred. Thus, for example, a polynucleotide that is at least 80% or 90% identical over 60, preferably over 100 nucleotides, as well as a polynucleotide that is at least 90% identical over 200 nucleotides, forms one aspect of the invention. .

  The BLAST N algorithm can be used to calculate sequence identity or enumerate sequences (eg, to identify sequences that are equivalent or corresponding to their default settings).

  Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm, when aligned with a word of the same length in a database sequence, has a short word of length W in the query sequence that either matches or satisfies several positively evaluated threshold scores T Is involved in first identifying a high scoring sequence pair (HSP). T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initializing searches to find HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. If the cumulative alignment score is reduced by an amount X from its maximum achieved value: if the cumulative score is 0 or less due to the accumulation of one or more negative scoring residue alignments; or at the end of any sequence If so, word hit expansion in each direction stops. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses 11 word lengths (W), 50 BLOSUM62 scoring matrix alignment (B), 10 expected values (E), M = 5, N = 4, and comparison of both strands as defaults .

  The BLAST algorithm performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)) that provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, the sequence has a minimum sum probability of comparing the first sequence to the second sequence of less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably about 0.00. If it is less than 001, it is considered similar to another sequence.

[Primers and probes]
The polynucleotides of the invention can be expressed by means of primers such as polymerase chain reaction (PCR) primers, primers for alternative amplification reactions, or conventional means using, for example, radioactive or non-radioactive labels. And may be used as a primer or probe as described above, or the polynucleotide may be cloned into a vector. Such primers, probes and other fragments are at least 15, such as at least 20, 25, 30 or 40 nucleotides in length. They are typically up to 40, 50, 60, 70, 100, 150, 200 or 300 nucleotides in length, or still a few nucleotides (eg, 5 or 10 nucleotides) shorter than the coding sequence of SEQ ID NO: 2. Up to length.

  In general, primers are generated by synthetic means, including stepwise production of the desired nucleic acid sequence one nucleotide at a time. Techniques and protocols for accomplishing this are readily available in the art. Longer polynucleotides are generally produced using recombinant means, eg, PCR cloning techniques. This creates a pair of primers (typically about 15-30 nucleotides) to amplify the desired region of the sialidase to be cloned, and the primer and yeast, bacteria, plant, prokaryotic cell or fungus Contact a cell, preferably mRNA, cDNA or genomic DNA obtained from a Penicillium strain, and perform a polymerase chain reaction under conditions suitable for amplification of the desired region (eg on an agarose gel). Isolating the amplified fragment (by purifying the reaction mixture) and recovering the amplified DNA. As described in Example 1, primers may be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate cloning vector.

  Alternatively, a synthetic gene can be constructed that includes the coding region of a secreted sialidase or variant thereof. Using these techniques, polynucleotides that have been altered at many positions but still encode the same protein can be conveniently designed and constructed. This has the advantage that the codon usage can be adapted to a suitable expression host and thus the protein production rate in this host can be improved. In addition, the polynucleotide sequence of the gene can be altered to improve mRNA stability or reduced turnover. This can result in improved expression of the desired protein or variant thereof. In addition, mutations are made in the protein sequence that have a positive effect on secretion efficiency, stability, vulnerability to proteolysis, optimal temperature, specific activity or other relevant properties for industrial production or use of the protein. As such, polynucleotide sequences can be altered in synthetic genes. In general, companies are available that provide services to construct synthetic genes and optimize codon usage.

  Such techniques may be used to obtain all or a portion of a polynucleotide encoding a sialidase sequence described herein. Introns, promoters and trailer regions are within the scope of the present invention, and also starting from genomic DNA from fungi, yeast, bacteria, plants or prokaryotic cells in a similar manner (eg, recombinant means, PCR or May be obtained by cloning techniques).

The polynucleotide or primer may carry an explicit label. Suitable labels include radioisotopes such as ³² P or ³⁵ S, fluorescent labels, enzyme labels or other protein labels such as biotin. Such labels can be added to the polynucleotides or primers of the invention and can be detected using techniques known to those skilled in the art.

  Labeled or unlabeled polynucleotides or primers (or fragments thereof) may be used in nucleic acid-based tests to detect or sequence sialidase or variants thereof in fungal samples. Such detection tests generally involve contacting a fungal sample suspected of containing the DNA of interest with a probe comprising a polynucleotide or primer of the invention under hybridizing conditions and And detecting any duplex formed between the nucleic acid and the nucleic acid in the sample. Detection uses techniques such as PCR, or the probe is immobilized on a solid support, any nucleic acid in the sample that does not hybridize to the probe is removed, and then any nucleic acid that hybridizes to the probe is removed. It can be achieved by detecting. Alternatively, sample nucleic acid may be immobilized on a solid support, the probe hybridized, and after removal of any unbound probe, the amount of probe bound to such support may be detected.

  The probe of the present invention may be conveniently packaged in a test kit in a suitable container. In such a kit, the probe may be bound to a solid support where the assay format for which the kit is designed requires such binding. The kit may also contain appropriate reagents, control reagents, instructions, etc. for processing the sample to be probed and for hybridizing the probe to the nucleic acid in the sample. The probes and polynucleotides of the invention may also be used in microassays.

  Preferably, the polynucleotide of the present invention is obtainable from the same organism as a polypeptide, such as a fungus, in particular a fungus of the genus Aspergillus.

[Polynucleotide production]
Polynucleotides that do not have 100% identity to SEQ ID NO: 2, but fall within the scope of the present invention, can be obtained in a number of ways. Therefore, variants of the sialidase sequences described herein can be obtained, for example, by searching a genomic DNA library made from a range of organisms as discussed as a source of the polypeptides of the invention. May be. In addition, other fungal, plant or prokaryotic homologues of sialidase can be obtained and such homologues and fragments thereof are generally capable of hybridizing to SEQ ID NO: 1. Such sequences can be used to search cDNA libraries or genomic DNA libraries from other species, and to identify such libraries with low, moderate to high stringency (as described above). Can be obtained by probing with a probe comprising all or part of SEQ ID NO: 1. Nucleic acid probes comprising all or part of SEQ ID NO: 1 may be used to search for cDNA or genomic libraries from other species as described as sources for the polypeptides of the invention.

  Species homologues may also be obtained using degenerate PCR using variants that encode conserved amino acid sequences and primers designed to target sequences within the homologue. A primer can contain one or more degenerate positions and is used at stringency conditions lower than those used to clone sequences with a single sequence primer against a known sequence. .

  Alternatively, such polynucleotides can be obtained by site-directed mutation of sialidase sequences or variants thereof. This can be useful, for example, when silent codon changes to the sequence are required to optimize codon selection for the particular host cell in which the polynucleotide sequence is expressed. Other sequence changes may be made to introduce restriction enzyme recognition sites or to alter the properties or functions of the polypeptide encoded by the polynucleotide.

  The present invention includes double-stranded polynucleotides comprising the polynucleotide of the present invention and its complement.

  The present invention also provides a polynucleotide encoding the above-described polypeptide of the present invention. This is generally desirable because such polynucleotides are useful for sequences for recombinant production of the polypeptides of the invention, but they hybridize to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Need not be possible. Otherwise, if desired, such polynucleotides may be labeled, used, and made as described above.

[Recombinant Polynucleotide]
The invention also includes vectors comprising a polynucleotide of the invention, including cloning and expression vectors, and in another embodiment, propagated, transformed, or encoded by, for example, a polypeptide of the invention, or a sequence of the invention. Methods are provided for transfecting such vectors into a suitable host cell under conditions that result in expression of the polypeptide to be produced. Also provided is a host cell comprising a polynucleotide or vector of the invention, wherein the polynucleotide is heterologous to the genome of the host cell. Generally, the term “heterologous” with respect to a host cell means that the polynucleotide does not occur naturally in the genome of the host cell or that the polypeptide is not naturally produced by the cell. Preferably, the host cell is a yeast cell, such as a yeast cell of the genus Kluyveromyces, Pichia, Hansenula or Saccharomyces, or, for example, Aspergillus, penicillium P A filamentous fungal cell of the genus Trichoderma or Fusarium.

[vector]
The vector into which the expression cassette of the present invention is inserted can be any vector that can be conveniently subjected to recombinant DNA procedures, and the choice of vector often depends on the host cell into which it is to be introduced. Therefore, the vector may be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity, such as a plasmid, and whose replication does not depend on chromosomal replication. Alternatively, a vector may be one that, when introduced into a host cell, is integrated into the genome of the host cell and replicates with the chromosome in which it is integrated.

  Preferably, when the polynucleotide of the invention is in a vector, it is operably linked to a regulatory sequence capable of providing expression of the coding sequence by the host cell (ie, the vector is an expression vector). ). The term “operably linked” refers to juxtaposition wherein the components described are in a relationship that allows them to function in their intended manner. Regulatory sequences such as promoters, enhancers or other expression control signals “operably linked” to the coding sequence are positioned in such a way that expression of the coding sequence is achieved under production conditions.

  The vector can be provided, for example, in the case of a plasmid, cosmid, virus or phage vector, with an origin of replication, optionally a promoter for expression of the polynucleotide, and optionally a promoter enhancer and / or regulator. A terminator sequence may be present and it may be a polyadenylation sequence. The vector may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of bacterial plasmids or a neomycin resistance gene in mammalian vectors. Vectors may be used in vitro, for example, for the production of RNA, or can be used to transfect or transform host cells.

  The DNA sequence encoding the polypeptide is preferably introduced into a suitable host as part of an expression construct in which the DNA sequence is operably linked to an expression signal capable of directing expression of the DNA sequence in the host cell. Is done. Transformation procedures well known to those skilled in the art are available for transformation of appropriate hosts with expression constructs. The expression construct can be used for host transformation as part of a vector carrying a selectable marker, or the expression construct is cotransformed as a separate molecule with a vector carrying a selectable marker. The vector may contain one or more selectable marker genes.

  Suitable selectable markers include, but are not limited to, those that compensate for defects in the host cell or confer resistance to drugs. They are, for example, acetamidase genes or cDNAs (amdS, niaD, facA genes or cDNAs from A. nidulans, A. oryzae, or A. niger) Includes multipurpose marker genes that can be used for transformation of most filamentous fungi and yeast, or genes that provide resistance to antibiotics such as G418, hygromycin, bleomycin, kanamycin, phleomycin or benomyl resistance (benA). Alternatively, specific selectable markers such as auxotrophic markers that require the corresponding mutant host strain: eg URA3 (a similar gene from S. cerevisiae or other yeast), pyrG or pyrA (A. nidulans or A. niger origin), argB (A. nidulans or A. niger origin) or trpC can be used . In a preferred embodiment, the selectable marker is transferred from the transformed host cell so that after introduction of the expression construct, a transformed host cell capable of producing a polypeptide free of the selectable marker gene is obtained. To be deleted.

  Other markers include ATP synthetase subunit 9 (oliC), orotidine-5′-phosphate-decarboxylase (pvrA), bacterial G418 resistance gene (useful in yeast but not in filamentous fungi), ampicillin resistance Examples include E. coli uidA gene encoding genes (E. coli), neomycin resistance gene (Bacillus) and glucuronidase (GUS). Vectors may be used in vitro, for example for the production of RNA or for transfecting or transforming host cells.

  For most filamentous fungi and yeast, the expression construct is preferably integrated into the genome of the host cell to obtain stable transformants. However, for certain yeasts, suitable episomal vector systems are also available that can incorporate expression constructs for stable and high levels of expression. Examples thereof include vectors derived from 2 μm of Saccharomyces and Kluyveromyces, CEN and pKD1 plasmids, respectively, or AMA sequences (eg, AMA1 from Aspergillus) Is mentioned. When integrating the expression construct into the host cell genome, the construct is either a random locus of the genome, or a predetermined target locus using homologous recombination (in this case, the target locus is preferably highly expressed). Or any other gene comprising a gene). A highly expressed gene is, for example, a gene whose mRNA can constitute at least 0.01% (w / w) of total cellular mRNA under induction conditions, or at least 0.2% of its gene product ( w / w) a total cellular protein or, in the case of a secreted gene product, a gene that can be secreted to a level of at least 0.05 g / l.

  Expression constructs for a given host cell are usually operably linked to each other in sequential order from the 5 'end to the 3' end to the coding strand of the sequence encoding the polypeptide of the first aspect. Containing the following elements: (1) a promoter sequence capable of directing transcription of the DNA sequence encoding the polypeptide in a given host cell, (2) preferably a 5 ′ untranslated region (leader), (3) optionally a signal sequence capable of directing secretion of a polypeptide from a given host cell into the culture medium, (4) a DNA sequence encoding a mature and preferably active polypeptide, and Preferably, (5) a transcription termination region (terminator) capable of terminating transcription downstream of the DNA sequence encoding the polypeptide.

  The expression construct downstream of the DNA sequence encoding the polypeptide preferably contains a 3 'untranslated region containing one or more transcription termination sites, also termed terminators. The origin of the terminator is not so important. The terminator can be derived, for example, from a DNA sequence encoding a polypeptide. Preferably, however, bacterial terminators are used in bacterial host cells, yeast terminators are used in yeast host cells, and filamentous fungal terminators are used in filamentous fungal host cells. More preferably, the terminator is endogenous to the host cell in which the DNA sequence encoding the polypeptide is expressed.

  Enhanced expression of a polynucleotide encoding a polypeptide of the present invention also increases expression of the protein of interest from the selected expression host, and, if desired, the level of secretion and / or It can also be achieved by selection of heterologous regulatory regions that serve to provide inducible control of peptide expression, such as promoters, signal sequences and terminator regions.

  In addition to the promoter derived from the gene encoding the polypeptide of the present invention, other promoters may be used to direct the expression of the polypeptide of the present invention. A promoter may be selected for its efficiency in directing expression of a polypeptide of the invention in the desired expression host.

  Promoters / enhancers and other expression control signals can be selected to be compatible with the host cell for which the expression vector is designed. For example, prokaryotic promoters, particularly promoters suitable for use in E. coli strains may be used. When expression of the polypeptide of the present invention is performed in mammalian cells, a mammalian promoter may be used. Tissue specific promoters, such as hepatocyte specific promoters, may be used. Use viral promoters such as Moloney murine leukemia virus long terminal repeat (MMLV LTR), Rous sarcoma virus (RSV) LTR promoter, SV40 promoter, human cytomegalovirus (CMV) IE promoter, herpes simplex virus promoter or adenovirus promoter You can also.

  Suitable yeast promoters include S. cerevisiae. S. cerevisiae GAL4 and ADH promoters and S. cerevisiae Examples include S. pombe nmt1 and adh promoter. Mammalian promoters include metallothionein promoters that can be induced in response to heavy metals such as cadmium. Viral promoters such as the SV40 large T antigen promoter or adenovirus promoter may be used. All these promoters are readily available in the art.

  Mammalian promoters such as the β-actin promoter may be used. Tissue specific promoters, particularly endothelial or nerve cell specific promoters (eg, DDAHI and DDAHII promoters) are particularly suitable. Viral promoters such as Moloney murine leukemia virus long terminal repeat (MMLV LTR), Rous sarcoma virus (RSV) LTR promoter, SV40 promoter, human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoter (eg HSV IE promoter) ), Or the HPV promoter, in particular the HPV upstream regulatory region (URR). Viral promoters are readily available in the art.

  A variety of promoters capable of directing transcription in the host cells of the invention can be used. Preferably, the promoter sequence is derived from a highly expressed gene as defined above. Preferably, triose-phosphate isomerase (TPI) is an example of a suitable highly expressed gene that is derived from a suitable predetermined target locus for promoter incorporation and / or for integration of an expression construct. Genes encoding glycolytic enzymes such as glyceraldehyde-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), pyruvate kinase (PYK), alcohol dehydrogenase (ADH), and amylase, glucoamylase, Examples include, but are not limited to, genes encoding proteases, xylanases, cellobiohydrolases, β-galactosidase, alcohol (methanol) oxidase, elongation factors and ribosomal proteins. Specific examples of suitable highly expressed genes include, for example, Kluyveromyces sp. LAC4 genes derived from Hansenula and Pichia, respectively, methanol oxidase genes (AOX and MOX), A. A. niger and A. niger A. awamori derived glucoamylase (glaA) gene, A. oryzae TAKA-amylase gene, A. awamori A. nidulans gpdA gene and T. An example is the T. reesei cellobiohydrolase gene.

  Examples of strong constitutive and / or inducible promoters suitable for use in fungal expression hosts include xylanase (xlnA), phytase, ATP-synthetase subunit 9 (oliC), triose phosphate isomerase (tpi), Promoters available from fungi for alcohol dehydrogenase (AdhA), amylase (amy), amyloglucosidase (AG- from glaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter There is.

  Examples of strong yeast promoters that can be used are available from the genes for alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, lactase, 3-phosphoglycerate kinase, plasma membrane ATPase (PMA1) and triose phosphate isomerase Promoters.

  Examples of strong bacterial promoters that can be used include amylase and SPo2 promoters and promoters from extracellular protease genes.

  Suitable promoters for plant cells that can be used include naparin synthase (nos), octopine synthase (ocs), mannopine synthase (mas), ribulose small subunit (rubisco ssu), histone, rice actin, phaseolin, Cauliflower mosaic virus (CMV) 35S and 19S and circovirus promoters.

  The vector may further comprise a sequence flanking the polynucleotide that yields an RNA comprising a sequence that is homologous to a eukaryotic genomic sequence, preferably a fungal genomic sequence, or a yeast genomic sequence. This allows the introduction of the polynucleotide of the invention into the genome of a fungus or yeast by homologous recombination. In particular, plasmid vectors comprising expression cassettes flanked by fungal sequences can be used to prepare vectors suitable for delivering the polynucleotides of the invention to fungal cells. Transformation techniques using these fungal vectors are known to those skilled in the art.

  The vector may contain a polynucleotide of the invention oriented in the antisense direction to provide for the production of antisense RNA. If desired, this may be used to reduce the level of expression of the polypeptide.

[Host cells and expression]
In a further embodiment, the present invention cultivates a host cell transformed or transfected with an expression vector as described above under conditions suitable for expression by the vector of a coding sequence encoding the polypeptide, and expresses the expressed polypeptide. A method for preparing a polypeptide of the invention comprising recovering The polynucleotide of the present invention can be incorporated into a replicable recombinant vector such as an expression vector. Vectors can be used to replicate nucleic acids in compatible host cells. Thus, in a further embodiment, the invention introduces a polynucleotide of the invention into a replicable vector, introduces the vector into a compatible host cell, and cultures the host cell under conditions that result in replication of the vector. Thus, a method for producing the polynucleotide of the present invention is provided. Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines, and other eukaryotic cell lines, eg insect cells such as Sf9 cells, and (eg filamentous) fungal cells. Is mentioned.

  Preferably, a polypeptide is produced as a secreted protein when the DNA sequence encoding the mature form of the polypeptide is operably linked to the DNA sequence encoding the signal sequence in the expression construct. If the gene encoding the secreted protein has a signal sequence in the wild type strain, preferably the signal sequence used is derived from (same as) the DNA sequence encoding the polypeptide. Alternatively, the signal sequence is foreign (heterologous) to the DNA sequence encoding the polypeptide, in which case the signal sequence is preferably endogenous to the host cell in which the DNA sequence is expressed. Examples of signal sequences suitable for yeast host cells include signal sequences derived from the yeast MFα gene. Similarly, suitable signal sequences for filamentous fungal host cells include, for example, the fungal amyloglucosidase (AG) gene, such as A. There is a signal sequence derived from the A. niger glaA gene. This signal sequence may be used in combination with the amyloglucosidase (also called (gluco) amylase) promoter itself, as well as in combination with other promoters. Hybrid signal sequences can also be used in connection with the present invention.

  Suitable heterologous secretory leader sequences include the fungal amyloglucosidase (AG) gene (glaA—both 18 and 24 amino acid versions from Aspergillus, for example), the MFα gene (yeast, eg, Saccharomyces, Pichia ( Pichia) and those derived from the Kluyveromyces) or α-amylase gene (Bacillus).

  The vector may be transformed or transfected into a suitable host cell as described above to provide for expression of the polypeptide of the invention. This process may comprise culturing host cells transformed with the expression vector as described above under conditions suitable for expression of the polypeptide, and optionally recovering the expressed polypeptide.

  Therefore, a further aspect of the invention provides a host cell transformed or transfected with a polynucleotide or vector of the invention or comprising a polynucleotide or vector of the invention. Preferably, the polynucleotide is carried in a vector that allows replication and expression of the polynucleotide. The cells are selected to be compatible with the vector and may be, for example, prokaryotic (eg, bacteria), or eukaryotic fungal, yeast, or plant cells.

  The invention includes a process for the production of a polypeptide of the invention by recombinant expression of a DNA sequence encoding a polynucleotide. For this purpose, the DNA sequences of the invention may be used for gene amplification and / or expression signals, such as promoters, secretion signals, in order to allow the economical production of the polypeptide in suitable homologous or heterologous host cells. Can be used for sequence exchange. Homologous host cells are defined herein as host cells that are the same species or are variants within the same species from which the DNA sequence is derived.

  Suitable host cells are preferably prokaryotic microorganisms such as bacteria, or more preferably eukaryotic organisms such as fungi such as yeasts or filamentous fungi, or plant cells. In general, yeast cells are preferred over filamentous fungal cells because yeast cells are easier to manipulate. However, some proteins are either poorly secreted from yeast or in some cases are not processed properly (eg, hyperglycosylation in yeast). In these cases, a filamentous fungal host organism should be selected.

  Bacteria from the genus Bacillus are highly suitable as heterologous hosts because of their ability to secrete proteins into the culture medium. Other bacteria suitable as hosts are those from the genus Streptomyces and Pseudomonas. Suitable yeast host cells for expression of the DNA sequence encoding the polypeptide are Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, or Schizo A member of the genus Schizosaccharomyces. More preferably, the yeast host cells are Saccharomyces cerevisiae, Kluyveromyces lactis (also known as Kluyveromyces marxius marxius marxius marxius marxians. ), Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica, and a group of species Schizosaccharomyces pombe (Schizosaccharomyces pombe).

  However, filamentous fungal host cells are most preferred for expression of the DNA sequence encoding the polypeptide. Suitable filamentous fungal host cells are Aspergillus, Trichoderma, Fusarium, Disporotrichum, Penicillium, Aurerosium, Aurerosium Selected from the group consisting of (Thermoascus), Myceliophtra, Sporotrichum, Thielavia, and Talaromyces. More preferably, the filamentous fungal host cell is a species of Aspergillus oyzae, Aspergillus sojae or Aspergillus nidulans (Aspergillus nidulans) or Aspergillus niger Raper and Fennel, The Genus Aspergillus, The Williams & Wilkins Company, Baltimore, pages 293-344, 1965). These include, Aspergillus niger (Aspergillus niger), Aspergillus awamori (Aspergillus awamori), Aspergillus tubigensis (Aspergillus tubigensis), Aspergillus Akureatasu (Aspergillus aculeatus), Aspergillus Foetidasu (Aspergillus foetidus), Aspergillus nidulans (Aspergillus nidulans ), Aspergillus japonicus, Aspergillus oryzae and Aspergillus ficuum, and also Trichoderma ree Trichoderma reesei, Fusarium graminearum, Penicillium chrysogenum, Acremonium acermos, Acremonium alabense. Cellulofilm (Sporotrichum cellulophilum), Disporotrichum dimorphosporum and Thielavia terrestr is) species, but is not limited to these.

  Examples of suitable expression hosts within the scope of the present invention include fungi such as Aspergillus species (particularly EP-A-184,438 and EP-A-284). And Trichoderma species; bacteria such as Bacillus species (particularly EP-A-134,048 and EP-A). 253,455), in particular, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Pseudomonas omonas species, as well as yeasts such as Kluyveromyces species (particularly EP-A-096,430, eg Kluyveromyces lactis) and European patents And those of Saccharomyces species, such as Saccharomyces cerevisiae, as described in published application A-301,670).

  Host cells according to the present invention include plant cells, and thus the present invention extends to transgenic organisms, such as plants and parts thereof, which contain one or more cells of the present invention. The cell may express the polypeptide of the invention heterologously or may contain one or more polynucleotides of the invention as heterologous. Thus, a transgenic (or genetically modified) plant can insert (typically stably) a sequence encoding a polypeptide of the invention into its genome. Plant cell transformation can be performed using known techniques, for example, using Ti or Ri plasmids derived from Agrobacterium tumefaciens. Therefore, the plasmid (or vector) may contain the sequences necessary to infect plants and Ti and / or Ri plasmid derivatives may be used.

  Host cells may overexpress the polypeptide, and techniques for manipulating overexpression are well known and can be used in the present invention. Therefore, the host may have 2 copies or more of the polynucleotide.

  Alternatively, direct infection of a part of a plant such as leaves, roots or stems can be performed. In this technique, a plant to be infected can be damaged by, for example, cutting the plant with a razor, puncturing the plant with a needle, or rubbing the plant with an abrasive. Next, Agrobacterium is seeded on the damaged part. The plant or plant part can then be grown on a suitable culture medium and grown into mature plants. Regeneration of transformed cells into genetically modified plants can be accomplished by selecting transformed shoots using known techniques, eg, antibiotics, and appropriate nutrients, plant hormones. It can be achieved by subculturing shoots on a medium containing the above.

[Culture and recombinant production of host cells]
The invention also includes cells that have been modified to express sialidase or a variant thereof. Such cells include transiently or preferably stably modified higher eukaryotic cell lines such as mammalian or insect cells, lower eukaryotic cells such as yeast and filamentous fungal cells, or prokaryotic cells, An example is bacterial cells.

  It is also possible to transiently express the polypeptide of the present invention on a cell line or a membrane such as a baculovirus expression system. Such systems adapted to express proteins according to the present invention are also included within the scope of the present invention.

  According to the present invention, the production of the polypeptide of the present invention can be carried out by culturing a microbial expression host, which is carried out in a conventional nutrient fermentation medium with one or more polynucleotides of the present invention. It has been transformed.

  Recombinant host cells according to the present invention can be cultured using procedures known to those skilled in the art. For each combination of promoter and host cell, culture conditions leading to expression of the DNA sequence encoding the polypeptide are available. After reaching the desired cell density or polypeptide titer, the culture is discontinued and the polypeptide is recovered using known procedures.

  Fermentation media include carbon sources (eg, glucose, maltose, molasses, etc.), nitrogen sources (eg, ammonium sulfate, ammonium nitrate, ammonium chloride, etc.), organic nitrogen sources (eg, yeast extract, malt extract, peptone, etc.) and inorganic It may comprise a known culture medium containing a nutrient source (eg, phosphate, magnesium, potassium, zinc, iron, etc.). Optionally, an inducer (depending on the expression construct used) may be included or added later.

  The selection of the appropriate medium may be based on the choice of expression host and / or based on the regulatory requirements of the expression construct. Suitable media are well known to those skilled in the art. The medium may contain additional components, if desired, that prefer the transformed expression host over other potentially contaminating microorganisms.

  The fermentation can be carried out over a period of 0.5-30 days. Fermentation can be a batch, continuous or fed-batch process at a temperature in the range between 0 ° C. and 45 ° C. and for example at a pH of 2 to 10. Suitable fermentation conditions include temperatures in the range between 20 ° C. and 37 ° C. and / or pH between 3-9. Appropriate conditions are usually selected based on the choice of expression host and the protein to be expressed.

  After fermentation, if necessary, the cells can be removed from the fermentation broth by centrifugation or filtration. After fermentation has stopped or after removal of the cells, the polypeptides of the invention can then be recovered and, if desired, purified and isolated by conventional means. The sialidase of the present invention can be purified from fungal mycelium or from a culture broth from which sialidase is released by cultured fungal cells.

  In a preferred embodiment, the polypeptide is produced from a fungus, more preferably Aspergillus, most preferably Aspergillus niger.

[Modification]
The polypeptide of the present invention may be chemically modified, for example, post-translationally modified. For example, they can be glycosylated (one or more times) or comprise modified amino acid residues. They may also be modified by the addition of histidine residues to aid their purification or by the addition of signal sequences to facilitate secretion from the cell. Polypeptides are amino terminal methionine residues, amino or carboxyl terminal extensions such as small linker peptides up to about 20-25 residues, or small extensions that facilitate purification such as polyhistidine tracts, antigenic epitopes or binding domains. You may have.

The polypeptide of the present invention may be labeled with an explicit label. The explicit label can be any suitable label that allows the polypeptide to be detected. Suitable labels include radioisotopes such as ¹²⁵ I, ³⁵ S, enzymes, antibodies, polynucleotides and linkers such as biotin.

  Polypeptides may contain non-naturally occurring amino acids or may be modified to increase the stability of the polypeptide. Where the protein or peptide is produced by synthetic means, such amino acids may be introduced during production. The protein or peptide may also be modified after synthetic or recombinant production.

  The polypeptides of the present invention may also be produced using D-amino acids. In such cases, the amino acids are linked in the reverse orientation in a C to N orientation. This is conventional in the art for producing such proteins or peptides.

Many side chain modifications are known in the art and can be made to the side chains of the proteins or peptides of the invention. Such modifications include, for example, reductive alkylation, reaction with aldehydes, followed by reduction with NaBH ₄ , modification of amino acids by amidation with methyl acetimidate or acylation with acetic anhydride.

  The sequences provided by the present invention may also be used as starting materials for the construction of “second generation” enzymes. A “second generation” sialidase is a sialidase modified by wild-type sialidase or a mutation technique (eg, site-directed mutagenesis or gene shuffling techniques) that has different properties from the recombinant sialidase as produced by the present invention. is there. For example, their temperature or optimum pH value, specific activity, substrate affinity or heat resistance may be altered to be better adapted for use in a particular process.

  The amino acids that are essential for the activity of the sialidase of the invention and are therefore preferably subjected to substitution may be identified according to procedures known to those skilled in the art, for example, site-directed mutagenesis or alanine-scanning mutagenesis. Good. In the latter technique, mutations are introduced at every residue in the molecule, and the resulting mutation is identified for biological activity (eg, sialidase activity) to identify amino acid residues that are critical to the activity of the molecule. To be tested. The site of enzyme-substrate interaction can also be determined by analysis of the crystal structure, as determined by techniques such as nuclear magnetic resonance, crystallography or photoaffinity labeling.

  Gene shuffling techniques provide a random method for introducing mutations into polynucleotide sequences. After expression, isolates with the best properties are reisolated, combined, and reshuffled to increase genetic diversity. By repeating this procedure many times, the gene encoding the improved protein can be rapidly isolated. Preferably, the gene shuffling procedure begins with a family of genes that encode proteins with similar functions. The family of polynucleotide sequences provided by the present invention is well adapted for gene shuffling to improve the properties of secreted sialidase.

  Alternatively, classical random mutagenesis techniques and selections such as NTG treatment or UV mutagenesis can be used to improve protein properties. Mutagenesis can be performed directly on isolated DNA or cells transformed with the DNA of interest. Alternatively, mutations can be introduced into isolated DNA by a number of techniques known to those skilled in the art. Examples of these methods include error-prone PCR, plasmid DNA amplification in repair-deficient host cells, and the like.

  The use of yeast and filamentous fungal host cells is expected to provide post-translational modifications such as proteolytic processing, myristylation, glycosylation, truncation, and tyrosine, serine or threonine phosphorylation. May be necessary to confer optimal biological activity on the recombinant expression product of the present invention.

[Preparation]
The polypeptides of the present invention can be in isolated form. It will be appreciated that the polypeptide may be mixed with a carrier or diluent that does not interfere with the intended purpose of the polypeptide and still be recognized as isolated. A polypeptide of the invention can also be in substantially purified form, in which case it generally exceeds 70% of the protein in a preparation that is a polypeptide of the invention, eg, 80% More than 90%, 95%, 98% or 99% of the polypeptide in the preparation.

  The polypeptides of the present invention can be provided in such a way that they exist outside their natural cellular environment. Therefore, they can be substantially isolated or purified as described above, or they can be present in cells that do not exist in nature, such as other fungal species, animals, plants or bacterial cells.

[Removal or reduction of sialidase activity]
The present invention also provides a method for generating a mutant cell of a parent cell comprising destroying or deleting an endogenous nucleic acid sequence encoding a polypeptide or its regulatory sequence, wherein the It relates to a method of producing mutant cells producing peptides.

  Construction of strains with reduced sialidase activity can be conveniently accomplished by modification or inactivation of the nucleic acid sequence required for sialidase expression in the cell. The nucleic acid sequence to be modified or inactivated can be, for example, a nucleic acid sequence encoding a polypeptide or a portion thereof essential to exhibit sialidase activity, or the nucleic acid sequence is derived from the coding sequence of the nucleic acid sequence. It may have a regulatory function necessary for expression of the polypeptide. An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, ie a part sufficient to influence the expression of the polypeptide. Other control sequences for possible modifications include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, signal sequences, and termination sequences.

  Modification or inactivation of the nucleic acid sequence can be accomplished by subjecting the cell to mutagenesis and selecting cells that have reduced or eliminated ability to produce sialidase. Mutagenesis, which may be specific or random, is, for example, by the use of suitable physical or chemical mutagens, by the use of suitable oligonucleotides, or by DNA sequence PCR mutagenesis. Can be implemented. In addition, mutagenesis can be performed through the use of any combination of these mutagens.

  Examples of physical or chemical mutagens suitable for this purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (NTG), O-methylhydroxylamine, Examples include nitric acid, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs.

  When using such factors, mutagenesis typically involves incubating the cells to be mutagenized in the presence of the preferred mutagen under appropriate conditions and reducing the expression of sialidase activity. Or by selecting cells that do not show expression.

  Altering or inactivating the production of a polypeptide of the invention is accomplished by introduction, substitution, or removal of one or more nucleotides in a nucleic acid sequence encoding the polypeptide, or a regulatory element necessary for its transcription or translation. Can be done. For example, nucleotides may be inserted or removed such that a stop codon is introduced, a start codon is removed, or an open reading frame is altered. Such modification or inactivation can be achieved by site-directed mutagenesis or PCR mutagenesis according to methods known in the art.

  In principle, the modification may be carried out in vivo, ie directly on the cell expressing the nucleic acid sequence to be modified, but the modification is preferably carried out in vitro as exemplified below.

  An example of a convenient way to inactivate or reduce the production of sialidase by a preferred host cell is based on the technique of gene replacement or gene interruption. For example, in a gene disruption method, a nucleic acid sequence corresponding to an endogenous gene or gene fragment of interest is mutagenized in vitro to produce a defective nucleic acid sequence and then transformed into a host cell to produce a defective gene. . By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene or gene fragment. Preferably, the defective gene or gene fragment also encodes a marker that can be used to select transformants in which the gene encoding the polypeptide has been altered or destroyed.

  Alternatively, modification or inactivation of a nucleic acid sequence encoding a polypeptide of the invention can be accomplished by established antisense techniques using nucleotide sequences that are complementary to the polypeptide coding sequence. More specifically, production of a polypeptide by a cell can be reduced or eliminated by introducing a nucleotide sequence that is complementary to a nucleic acid sequence encoding the polypeptide. The antisense polynucleotide is then typically transcribed in the cell and is capable of hybridizing to mRNA encoding sialidase. Under conditions where a complementary antisense nucleotide sequence is hybridized to the mRNA, the amount of sialidase produced in the cell is reduced or eliminated.

  The cells to be modified according to the method of the present invention are preferably derived from a microorganism, eg, a fungal strain that is suitable for production of the desired protein product that is either homologous or heterologous to the cells.

  The present invention further relates to a parental cell mutant cell comprising a disruption or deletion of an endogenous nucleic acid sequence encoding the polypeptide or its regulatory sequence, resulting in a mutant cell that produces less polypeptide than the parent cell.

  Polypeptide-deficient mutant cells so produced are particularly useful as host cells for the expression of homologous and / or heterologous polypeptides. Accordingly, the present invention further produces a homologous or heterologous polypeptide comprising (a) culturing the mutant cell under conditions that lead to the production of the polypeptide; and (b) recovering the polypeptide. Related to the method. In this regard, the term “heterologous polypeptide” as used herein refers to a polypeptide that is not derived from a host cell, a native protein that has been modified to alter its native sequence, or a host cell that is expressed by recombinant DNA technology. Is defined as a natural protein that is quantitatively altered as a result of the manipulation of

  In yet a further aspect, the present invention provides a method for producing a protein product essentially free of sialidase activity by fermentation of cells that produce both the sialidase polypeptide of the invention as well as the protein product of interest. The method adds an effective amount of an agent capable of inhibiting sialidase activity to the fermentation broth, either during the fermentation or after the fermentation is complete, recovers the desired product from the fermentation broth, and Subjecting the recovered product to further purification. Alternatively, after culturing, the resulting culture broth can be subjected to a pH or temperature treatment to substantially reduce sialidase activity and allow recovery of the product from the culture broth. A combined pH or temperature treatment may be performed on the protein preparation recovered from the culture broth.

  The method of the present invention for producing a product that is essentially free of sialidase is particularly interesting in the production of eukaryotic polypeptides, particularly in the production of fungal proteins such as enzymes. Sialidase-deficient cells may also be used to express a heterologous protein of interest for the food industry or pharmaceutical purposes.

[Cheese making and milk coagulation]
Coagulation is an essential step in the traditional production of cheese from dairy compositions such as milk. Clotting can be initiated by acidification and / or addition of an enzyme (coagulant) such as chymosin. After coagulation, the milk is separated into curd and whey. The curd is further processed into cheese.

The cheese making process from various milk sources has been known for quite some time and has been described in detail for many different types of cheese variants. (E.g., Cheese: Chemistry, Physics and Microbiology, Volumes 1 and 2, 1999, Fox Edition, Aspen Publications, Gaithersburg, Maryland; Encyclopedia of A4, A4 (See Press, London). A very important point in cheese making is the process of coagulation, where the solubility of casein micelles and submicelles is reduced. Enzyme-induced coagulation is very commonly used. Enzymes such as calf chymosin, microbial equivalents of chymosin, and other enzymes from other sources have also been described, and some are available under various trade names. All of them can be used to initiate the coagulation process. The primary step of clotting is cleavage of the Phe ₁₀₅ -Met ₁₀₆ bond in κ-casein. This results in removal of the C-terminal part of κ-casein: glycomacropeptide (GMP). Removal of GMP leads to casein micelle association, ie, casein coagulation. Casein coagulation results in gel formation and the time required to obtain gelation in a particular dairy composition is directly related to the activity of the coagulant.

  The time that elapses between the addition of the coagulant and the appearance of the initial casein aggregate is defined as the coagulation time. The rate at which gels are formed in cheese milk and the density of the gels are closely dependent on the amount of enzyme added, the concentration of calcium ions, phosphorus, temperature and pH. After initial solidification, a gel forms and the consistency of the gel increases with increasing intermicellar binding. The micelles move together and the clot shrinks, thereby releasing whey. This phenomenon is known as syneresis and is accelerated by cutting the card, increasing the temperature and increasing the acidity caused by the growing lactic acid bacteria.

  The time between the addition of the coagulant and the cutting of the gel for initiating liquid separation is hereinafter referred to as the coagulation time. For example, minor variations (up to 5-10%) can occur as a result of changes in milk or coagulant quality, but the clotting time is constant for a particular cheese type in a particular cheese factory. In many cheese factories, clotting time is a time limiting process in the process. If the clotting time decreases, the factory can process more milk into curd, then tan, and then further into cheese. Therefore, the reduction of clotting time is economically interesting and there is a need for such a reduction of clotting time.

  Coagulation time can be reduced in several ways, such as increasing the coagulant added, lowering the pH of the milk, or increasing the level of calcium chloride added. However, these solutions have not been widely used in the cheese making industry because they negatively affect cheese quality. Increasing the level of coagulant often results in the formation of bitter taste as a result of unbalanced protease activity. Decreasing milk pH results in poor quality curd and often results in yield loss. Increases in calcium chloride concentration have a negative impact on taste and are often legally limited. There are no currently available methods for reducing clotting time without negative side effects.

  The stability of casein micelles against aggregation is critical in the aggregation process. κ-casein plays an extremely important role in casein micelle stabilization. The hydrophilic and negatively charged C-terminal part of the κ-casein chain localized on the micelle surface causes steric hindrance and electrostatic repulsion, and prevents the clotting of micellar casein (Minkiewickz) , Et al., Pol J Food Nutr Sci (1993) 243, 39-48). Kappa-casein is a glycoprotein containing sialic acid residues at known positions (Cases et al., J Food Sci (2003) 68, 2406-2410; Fournet et al., Biochim Biophys Acta ( (1979) 576, 339-346). Based on glycosylation differences and sialic acid content, it is known that micro-heterogeneity exists in populations of kappa-casein (Robitaille et al., Food Res Int (1995) 28, 17-21; Robitaille et al., J Dairy Res (1991) 58, 107-114). Several studies have described the effect of removing sialic acid residues using sialidase from Clostridium perfringens. The parameter examined is the stability of casein micelles against heating and chymosin degradation. Gibbons et al. (Biochim Biophys Acta (1962) 56, 354-356) used a solution of purified kappa-casein and the removal of sialic acid residues contributed to the action of chymosin on kappa-casein. It showed no effect. Vreeman et al. (Biochem J (1986) 240, 87-97) compare the kinetic behavior of chymosin to purified κ-casein fractions with and without glycosylation and deglycosylation. We have found that κ-casein is a better substrate for chymosin. In addition, Minkiewicz et al. (Pol J Food Nutr Sci (1993), 243, 39-48) uses an artificially reconstituted casein micelle system in which sialic acid residues are converted into casein. It has been shown to contribute to the heat resistance of micelles, which was later confirmed by Robitaille et al. (Food Res Int (1995) 28, 17-21). In a separate study, Robitail et al. (Food Res Int (1993) 26, 365-369) showed that removal of sialic acid residues from κ-casein did not significantly affect clotting time, The card's firmness has been reduced. There is no description of a cheese making process with a reduction in clotting time but without affecting the properties of the cheese, and this is an industry need.

  A recent patent application (European Patent No. 1370146) describes a process for coagulating milk using a single glycosidase containing N-acetylneuraminidase. In the described configuration, no enzyme coagulant is used. While the omission of coagulants opens the way to alternative cheese making processes, (proteolytic) coagulants are an important part of the proteolytic system in cheese and the formation of characteristic flavor compounds Is not an attractive process for traditional cheese making processes (see, for example, Cheese: Chemistry, Physics and Microbiology, Vols. 1 and 2, 1999, for references on the role of chymosin in cheese flavor formation) See Fox, Aspen Publications, Gaithersburg, Maryland; Encyclopedia of Daily Sciences, Volumes 1-4, 2003, Academic Press, London. )

  In this regard, the term “cheese” refers to any type of cheese such as, for example, natural cheese, cheese analogs and processed cheese. Cheese is any suitable process known in the art such as, for example, by enzymatic coagulation of dairy compositions with rennet, or acid coagulation of dairy compositions with food grade acids or acids produced by lactic acid bacteria growth. Can be obtained by: In one embodiment, the cheese is rennet-curd cheese. The dairy composition can be subjected to a conventional cheese making process. Process cheese is preferably made from natural cheese or cheese analogs by cooking and emulsifying the cheese with, for example, emulsifying salts (eg, phosphate and citrate). The process may further include the addition of spices / condiments.

  The term “cheese analogue” includes fat as part of the composition (eg, milk fat (eg, cream)) and as part of one composition, eg, non-milk components such as vegetable oil. Further refers to cheese-like products that contain cheese, which can comprise all kinds of cheeses such as soft cheeses, semi-hard cheeses and hard cheeses. , Rennet-curd and acid-curd cheese, either by rennet or acidification alone, is performed by means of fresh acid-curd cheese via milk, cream or A type of cheese that is produced by whey coagulation and that is ready to be consumed without ripening once production is complete. When a high temperature is used, coagulation usually occurs around the isoelectric point of casein, i.e., around a pH of 4.6 or higher (e.g., typically about 6.0 in Ricotta). In terms of pH, and typically at a temperature of about 80 ° C., fresh acid-curd cheese is generally a type of rennet-curd cheese (eg, Camebert, Cheddar, Emmental). Here, clotting is usually induced by the action of rennet at a pH value of 6.4 to 6.6.In a preferred embodiment, the cheese belongs to the class of rennet curd cheese.

  The dairy composition can be any composition comprising a milk component. The milk component can be any component of milk such as milk fat, milk protein, casein, whey protein and lactose. The milk fraction can be any fraction of milk such as, for example, skim milk, butter milk, whey, cream, milk powder, whole milk powder, skim milk powder. In a preferred embodiment, the dairy composition comprises milk, skim milk, buttermilk, whole milk, whey, cream, or any combination thereof. In a more preferred embodiment, the dairy composition consists of milk such as skim milk, whole milk, cream or any combination thereof. In a further embodiment, the dairy composition is prepared in whole or in part from a dry milk fraction such as, for example, whole milk powder, skim milk powder, casein, casein salt, total milk protein or sweetened milk powder, or any combination thereof. Is done. The dairy composition comprises milk and / or one or more milk fractions. The milk fraction can be from any breed of cattle (Bos taurus taurus, Bob indicus taurus) and their crosses. In one embodiment, the dairy composition comprises two or more cattle The dairy composition also contains milk from other mammals used in cheese preparations such as goat, buffalo or camel milk. A dairy composition for the preparation of cheese is normalized to the desired composition by removal of all or part of the raw milk ingredients and / or by adding further amounts of such ingredients to it. This can be done, for example, by separating milk into cream and milk upon arrival at the dairy. The dairy composition can be prepared as conveniently done by fractionating the milk and recombining the fractions so that the desired final composition of the dairy composition is obtained. It may be performed in a continuous centrifuge, resulting in a skimmed milk fraction with a very low fat content (ie <0.5%) and for example a cream with> 35% fat. In another embodiment, the protein and / or casein content can be standardized by the use of ultrafiltration The dairy composition is the cheese to be produced by the process of the present invention. May have any total fat content found to be suitable.

Calcium can be added to the dairy composition at any suitable stage before and / or during cheese making, such as before, simultaneously with, or after the starter culture is added. In a preferred embodiment, calcium is added both before and after the heat treatment. Calcium may be added in any suitable form. In a preferred embodiment, calcium, calcium salt, for example, is added as CaCl _2. Any suitable amount of calcium can be added to the dairy composition. The concentration of calcium added is usually in the range of 0.1 to 5.0 mM, such as between 1 and 3 mM. When adding CaCl ₂ to the dairy composition, the amount is usually in the range of 1-50 g per 100 liter dairy composition, for example in the range of 5-30 g per 1000 liter dairy composition, preferably 100 liter dairy composition It is in the range of 10 to 20 g per thing.

  The bacterial content of skim milk can be reduced by conventional preferences. The dairy composition may be subjected to a homogenization process prior to cheese production, as in the production of Danish Blue Cheese.

FIG. 1 shows the construction of a ZJW expression vector.

  The following examples illustrate the invention.

[Example]
[Example 1]
[Cloning and expression of sialidase gene ZJW]
Penicillium chrysogenum strain Wisconsin 54-1255 (ATCC 28089) was grown in PDB (potato dextrose broth, Difco) for 3 days at 30 ° C. and chromosomal DNA was used using the supplier's instructions, Isolation from the mycelium using the Q-Biogene kit (Catalog No. 6540-600; Omnilabo International BV, Breda, Netherlands). Chromosomal DNA was used to amplify the coding sequence of the sialidase gene using PCR.

Two PCR primers were designed to specifically amplify the sialidase gene ZJW from the chromosomal DNA of Penicillium chrysogenum strain Wisconsin 54-1255 (ATCC 28089). The primer sequence was partially obtained from the sequence found in the genomic DNA of Penicillium chrysogenum Wisconsin 54-1255 (ATCC 28089) and is shown in SEQ ID NO: 1. We have found that this sequence has homology with the Actinomyces and Arthrobacter sialidase sequences. However, no description of homologous fungal sialidase has yet been observed. Therefore, it is surprising that the inventors were able to find a gene encoding a secreted sialidase from a fungus. We describe here for the first time the efficient expression and characterization of secreted fungal sialidase. The protein sequence of the complete sialidase protein comprising potential pre- and pro-sequences is shown in SEQ ID NO: 3. The advantage of a fungal enzyme compared to a bacterial homolog is that the fungal enzyme is easily overexpressed and can be secreted in amounts relevant for application in the food industry.

  The first direct PCR primer (ZJW-dir) contains a 23 nucleotide ZJW coding sequence starting from the ATG start codon and a 23 nucleotide sequence including a PacI restriction site in front of it (SEQ ID NO: 4). The second reverse primer (ZJW-rev) contains a nucleotide complementary to the reverse strand in the region downstream of the ZJW coding sequence and an AscI restriction site in front of it (SEQ ID NO: 5). Using these primers, we were able to amplify a 1.4 kb sized fragment with chromosomal DNA from Penicillium chrysogenum strain Wisconsin 54-1255 (ATCC 28089) as a template. . The 1.4 kb sized fragment thus obtained was isolated, digested with PacI and AscI and purified. The PacI / AscI fragment comprising the ZJW coding sequence was replaced with a PacI / AscI phyA fragment derived from pGBFIN-5 (WO99 / 32617). The resulting plasmid is a ZJW expression vector designated pGBFINZJW (see FIG. 1). The expression vector pGBFINZJW was linearized by digestion with NotI (this removes all E. coli derived sequences from the expression vector). The digested DNA was purified using phenol: chloroform: isoamyl alcohol (24: 23: 1) extraction and precipitated with ethanol. These vectors were used to transform Aspergillus niger CBS513.88. The Aspergillus niger transformation procedure is extensively described in WO 98/46772. It has also been described how to select transformants on agar plates containing acetamide and select targeted multicopy integrants. Preferably, A. containing multiple copies of the expression cassette. A. niger transformants are selected for further production of sample material. For the pGBFINZJW expression vector, the individual transformants were first plated on selective media, followed by plating a single colony on a PDA (potato dextrose agar: PDB + 1.5% agar) plate. A. Niger (A. niger) transformants were purified. After one week of growth at 30 ° C., spores of individual transformants were collected. Spores were stored in a refrigerator and used for seeding of liquid medium.

A. containing multiple copies of expression cassette A. niger strain was used to make the sample material by culturing the strain in shake flask culture. A. A useful method for culturing A. niger strains and separating mycelium from the culture broth is also described in WO 98/46772. The culture medium, CSM-MES (1 liters medium per 150g maltose, 60 g of Soytone (Difco), 15g of _{(NH 4) 2 SO 4,} 1g of ^{_{NaH 2 PO 4 · H 2 O}} , MgSO of 1 g ₄ ^· 7H ₂ O, 1 g L-arginine, 80 mg Tween-80, 20 g MES, pH 6.2). A 5 ml sample was taken on days 4-8 of the fermentation, centrifuged for 10 minutes at 5000 rpm in Hereaus RF, and the supernatant was stored at -20 ° C until further analysis.

  SDS-Page molecular weight (MW) determination: Analyze samples by SDS-PAGE on NuPAGE Novex Bis-Tris 4-12% gradient system (Invitrogen (Breda)) using NuPAGE MES-SDS running buffer To do. Reduce the sample prior to filling using NuPAGE reducing agent according to the supplier's protocol. To estimate the molecular weight of the protein produced, a marker protein (SeeBlue Plus2Pre-Stained Standard (Invitrogen)) is used. Gels were run and stained using Simply Blue Protein Strain (Invitrogen) according to the supplier's instructions, and the molecular weight of the protein produced was estimated by comparison with a standard protein. In this example, 13 microliters of supernatant of the transformant fermentation broth was analyzed.

  When analyzed by SDS-PAGE, it was found that the transformant containing the pGBFINZJW vector secreted a protein with an apparent molecular weight of approximately 50 kDa. Since this is slightly larger than the molecular weight deduced from the protein sequence, we have removed the signal sequence and if Penicillium chrysogenum sialidase ZJW is secreted from Aspergillus niger It is estimated that some glycosylation occurs.

  If fermentation and downstream processing are scaled up, the selected strain can be used for the isolation and purification of larger amounts of fungal sialidase. This enzyme can then be used for further analysis and use in a variety of individual applications.

[Example 2]
[Purification and characterization of sialidase]
Sialidase was produced via fermentation as described in Example 1. Enzyme activity was measured using the Amplex Red neuraminidase assay kit (obtained from Invitrogen). Culture filtrate (100 ml) was diluted with milliQ-water to a conductivity of 4.8 mS / cm and concentrated to 70 ml by ultrafiltration using a Biomax-10 membrane (obtained from Millipore). The pH was adjusted to 6.0 using NaOH and the sample was loaded onto a 5 ml HiTrapQ ion exchange column (5 ml / min obtained from Amersham) and in 20 mM sodium citrate (pH 6.0). Equilibrated. The flow through of the column containing sialidase is collected and dialyzed against 25 mM Tris, HCl (pH 7.0) and loaded onto 5 ml HiTrap Q FF (5 ml / min) and equilibrated in the same buffer. Turned into. Sialidase was present in the flow-through fraction and was recovered. The enzyme solution was then dialyzed against 30 mM sodium citrate (pH 4.0, buffer A) and applied onto a 5 ml HiTrap SP column (5 ml / min obtained from Amersham) and equilibrated in buffer A. Turned into. After loading the enzyme, the column was washed with 3 column volumes of buffer A and the enzyme was washed in a linear gradient from 20 column volumes of buffer A to buffer B (buffer B: 30 mM sodium containing 1 M NaCl. -Elution with citric acid, pH 4.0). Sialidase-containing fractions were identified and pooled. Protein concentration was determined by Bradford reagent (obtained from Sigma) using bovine serum albumin as a control protein. The protein was judged to be> 95% pure because no contaminating bands were observed on sodium-dodecyl polyacrylamide gel electrophoresis. Sialidase migrates to an apparent molecular weight of 47 kD, which is slightly larger than the 42.7 kD molecular weight calculated based on the deduced amino acid sequence. The enzyme preparation does not show proteolytic activity against a series of substrates ZAAXpNA (Z = benzoyl group, A = alanine, X = any amino acid residue, pNA = para-paranitroanilide) and has endo-protease activity. Indicates absence.

[Example 3]
[Preparation method of miniature cheese]
Miniature cheese was produced as described by Shakeel-Ur-Rehman et al. (Protocol for the manufacturer of miniature cheeses, Lait, 78, (1998), 607-620). Although pasteurized whole fat homogenized milk was used, raw milk or reduced milk can also be used. The milk was transferred to a wide-mouth plastic bottle (200 mL per bottle) and cooled to 31 ° C. Subsequently, 1.8 units of starter culture DS 5LT1 (DSM Gist BV, Delft, Netherlands) is added to each 200 ml of pasteurized milk in a centrifuge bottle and the milk is allowed to mature for 20 minutes. It was. Then CaCl ₂ (132 μL of ¹ mol.L ⁻¹ solution per 200 mL fermented milk) was added. Finally, a coagulant (0.04 IMCU per ml) was added. If sialidase was used, it was added with the coagulant. The milk solution was held at 31 ° C. for 40-50 minutes until a clot was formed. The agglomerates were manually cut with a wire cutter stretched over the frame at 1 cm intervals. It was allowed to recover for 2 minutes, followed by gentle stirring for 10 minutes. The temperature was then gradually increased to 39 ° C. for 30 minutes under continuous stirring of the curd / whey mixture. When a pH of 6.2 was reached, the curd / whey mixture was centrifuged at 1,700 g for 60 minutes at room temperature. The whey was drained and the curd was kept at 36 ° C. in a water bath. The cheese was inverted every 15 minutes until the pH dropped to 5.2-5.3 and then centrifuged at 1,700 g for 20 minutes at room temperature. After production, the cheese was weighed.

[Example 4]
[Method for determining cheese coagulation]
Optigraph (Alliance Instruments, France) was used to track cheese coagulation as described by Kubarsep et al. (Ata Agric Scan Section A (2005) 55, 145-148). Following the addition of the curd enzyme solution, the clotting time of the standard milk substrate solution was determined according to standard procedures (IDF157 A [2]). 11 g of low heat nonfat dry milk (Nilac, NIZO, was obtained from The Netherlands), by adding the milliQ- 100ml water containing CaCl ₂ of 4.5 mM, were prepared milk substrate solution. The milk solution was stirred with a magnetic stirrer for 30 minutes. The solution was placed in the dark for 30 minutes at room temperature to stabilize the milk. The pH of the milk is about 6.5. In order to achieve a rennet clotting time (r) of between 8 and 15 minutes, the curdling enzyme was diluted with milli-Q water. The 10 ml milk solution was transferred to an Optigraph sample cuvette. The milk was adjusted to a reaction temperature of 32 ° C. 200 μl of diluted enzyme sample was pipetted into a multiple spoon apparatus and added to the milk in one shot. This operation is the starting point of the coagulation test. The enzyme and milk were mixed with a spoon and supplied with Optigraph for this purpose. Clotting was monitored for 45 minutes.

Claims (18)

  Having amino acid sequence having sialidase activity and having at least 90% amino acid sequence identity with amino acids 1 to 407 of SEQ ID NO: 3 or having at least 90% amino acid sequence identity with amino acids 34 to 407 of SEQ ID NO: 3 An isolated polypeptide having an amino acid sequence.   2. The polypeptide of claim 1, wherein the polypeptide has a MW (Molecular Weight) (SDS-Page) of less than 55 kDa.   The polypeptide of claim 1 which is an extracellular enzyme.   The polypeptide according to any one of claims 1 to 3, which has an amino acid sequence having at least 95% identity with amino acids 1 to 407 of SEQ ID NO: 3 or amino acids 34 to 407 of SEQ ID NO: 3.   The polypeptide according to any one of claims 1 to 3, which has an amino acid sequence having at least 97% identity with amino acids 1 to 407 of SEQ ID NO: 3 or amino acids 34 to 407 of SEQ ID NO: 3.   The polypeptide of claim 1 comprising the amino acid sequence of SEQ ID NO: 3.   The polypeptide according to any one of claims 1 to 6, obtained from a fungus.   The polypeptide according to any one of claims 1 to 6, which is obtained from Penicillium.   The polypeptide according to any one of claims 1 to 6, obtained from Penicillium chrysogenum.   An isolated polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of claims 1-9.   11. A nucleic acid construct comprising the polynucleotide of claim 10 operably linked to one or more control sequences that direct the production of the polypeptide in a suitable expression host.   A recombinant expression vector comprising the nucleic acid construct of claim 11.   A recombinant host cell comprising the recombinant expression vector of claim 12 or the nucleic acid construct of claim 11.   10. The recombinant host cell of claim 13 is cultured to produce a supernatant and / or cell comprising the polypeptide; and the polypeptide is recovered. A method for producing the polypeptide of claim 1.   A polypeptide obtainable by the method of claim 14.   A method for producing the polypeptide according to any one of claims 1 to 9, comprising a polynucleotide encoding the polypeptide under conditions suitable for the production of the polypeptide. Culturing a host cell comprising; and recovering said polypeptide.   A polypeptide produced by the method of claim 16. Of a polypeptide according to any one of claims 1～9,15 and 17, use that put in the preparation of food or feed.

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